| Objective:1.Using bioinformatics technology to predict the T and B cell epitopes of Eg EF-1 and Eg TPI,in order to predict the structure,understand its diagnostic value.2.Constructing these two genes expression vector by using prokaryotic expression technology to obtain recombinant protein,which can lay the foundation for the subsequent research of immune protection.Methods: 1.Physicochemical property of Eg EF-1 was predicted using Expasy system.Hydrophiloity plot,flexible regions,antigenic index and surface accessibility were analyzed by DNAStar software.B cell linear antigen epitopes were forecasted inregratly by ABCpred and IEDB software.T cell antigen epitopes were forecasted by DNAStar software.The secondary and three-dimensional structure of Eg EF-1 were determined using the SOPMA software and SWISS-MODEL website separately.Compared the amino acid sequence of EF-1 and TPI in Eg and human respectively by using DNAstar software.2.The target gene,getting by RT-PCR,were connected to the cloning vector to form a recombinant plasmid.Then connected the later and Ndel/Xhol to form prokaryotic expression recombinant plasmid.After that,expressed and purified protein products.The serum Ig G were determined in 37 cases of CE,29 cases of AE and 16 cases of normal respectively with ELISA method to investigate the antigenicity and diagnostic of these two proteins.Results: The high score areas of hydrophiloity plot had seven.Six of flexible regions,The high score regions of antigenic index were similar with flexible regions.Surface accessibility had few high score regions.The potential amino acids sequence of B cell linear epitopes were 120-130? 286-295 ? 306-320 ? 365-378.The potential advantaged T cell epitope areas were 34-48,164-177,222-244,280-290,321-330,396-407.?-helix accounted for 32.81%,?-sheet accounted for 22.54%,?-corner accounted for 10.94%,and random coil accounted for 33.71% in the secondary structure of Eg EF-1.The results showed that the Eg EF-1 has a sequence consistency of 35.06% with the human EF-1,and the Eg TPI has a sequence consistency of 8% with the human TPI.The high score areas of Eg TPI hydrophiloity plot had eight.The high score regions of antigenic index were similar with flexible regions,Surface accessibility had few high score regions.The potential amino acids sequence of B cell linear epitopes were 28-39?50-60?70-80?131-139?150-159?213-231.The potential advantaged T cell epitope areas were 16-30?71-85?98-119?142-154?178-200.?-helix accounted for 36% ?-sheet accounted for 22.00%,?-corner accounted for 12.00%,and random coil accounted for 30.00%.2.After the construction of recombinant expression vector and the induction and purification of recombinant protein,we found that the molecular weight of Eg EF-1 and Eg TPI were about 34 k D and 27 k D respectively.According to the results of ELISA method,the detection rate of Eg EF-1 were negative with generally high OD values;the positive detection rate of Eg TPI in CE and AE were 86.48% and 72.41%.Compared with healthy people,the difference was statistically significant(P< 0.05),but there was no significant difference between CE and AE?Conclusion :1.Four B cell epitopes and six advanted T cell epiopes of Eg EF-1,six B cell epitopes and five advanted T cell epiopes of Eg TPI were acquired by bioinformatics methods.Eg EF-1 had high conservative according to the phylogenetic tree.These findings could provide basis for antigenicity research,immune diagnosis and drug treatment of them in Echinococcus granulous.2.After the successful expression of recombinant proteins,we conclude that the detection rate of EF-1 in two kinds of patients serum are negative;the results of Eg TPI testing showed that there are cross reaction between the CE and the AE,the recombinant protein(Eg TPI)has no genetically specific. |
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